The determination of the calcium content of various aqueous liquids is important for many manufacturing and environmental purposes. For example, it is often necessary to know the amount of calcium in wastewater, groundwater, food products, chemical processing fluids, oil well water or other natural or effluent liquid sources.
It is also important for diagnostic purposes to determine the calcium content in biological fluids. The calcium level is normally relatively constant in most body fluids. Change in calcium concentration in blood serum can indicate several pathological conditions. For example, hypercalcemia occurs in the hyperfunction of the parathyroid glands and the thyroid glands, in sarcoidosis and in several metastasizing carcinomas. Hypercalcemia is also connected with osteoporosis and osteoplastic carcinomas, acute pancreatitis and acidosis.
A number of chemical and physical procedures are known for the determination of calcium. Direct colorimetric procedures are preferred over tedious precipitation, gravimetric or titrimetric procedures. Generally, such colorimetric procedures involve the complexation of a dye with calcium ions to provide a measurable shift in dye absorption. Several colorimetric assays are described in U.S. Pat. Nos. 3,754,865 (issued Aug. 28, 1973 to Gindler) and 3,798,000 (issued Mar. 19, 1974 to Helger).
A potential problem in the determination of calcium ions in most fluids is the presence of potentially interfering ions (for example, magnesium or phosphates) or large molecules (for example, proteins or bilirubin). Magnesium ions present a particularly difficult problem because they tend to complex to the same compounds that complex with calcium ions. According to the art noted above, magnesium ions can be removed physically from the test sample prior to calcium determination or masked with a magnesium-specific complexing reagent.
A relatively recent advance in clinical chemistry was the development of thin-film multilayer analytical elements such as those described in U.S. Pat. Nos. 3,992,158 (issued Nov. 16, 1976 to Przybylowicz et al) and 4,258,001 (issued Mar. 24, 1981 to Pierce et al). These elements generally have a porous spreading layer and a reagent layer on a nonporous support. One such element has been designed for the determination of calcium ions and is marketed as a KODAK EKTACHEM Clinical Chemistry Slide by Eastman Kodak Co. (Rochester N.Y.). This element contains an arsenazo dye which selectively complexes with calcium ions to provide a detectable dye shift. However, environmental concerns regarding arsenic have prompted workers in the art to find new calcium complexing compounds.
Highly selective compounds for calcium ions are described by Tsien in Biochem., 19, pp. 2396-2404 (1980). The parent compound described therein is 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, commonly known as BAPTA. These compounds suffer from the disadvantage, however, that they absorb in the ultraviolet region of the electromagnetic spectrum. However, other species, such as bilirubin, hemoglobin and other porphorin species, and metabolic by-products of porphorins, found in analyte solutions such as blood plasma, spinal fluid, urine and other body fluids, also absorb in the UV and short visible wavelength portions of the electromagnetic spectrum, and produce background interference with standard colorimetric equipment and procedures. Therefore, it would be desirable to have highly selective calcium complexing compounds which would be detectable at longer wavelengths, and which would shift to other wavelengths when complexed with calcium to allow quantitative analysis for calcium without interference from UV and short wavelength visible light-absorbing species.